The molecular systems underlying complex traits are in general poorly understood. Even less well understood is how genetic and environmental differences between individuals translate into phenotypic differences. A general feature of this mapping from genotype to phenotype is robustness, or the buffering of the phenotype against genetic and environmental variation. Complex human diseases can be viewed as failures of robust systems, with phenotypic variation manifesting as individual differences in clinical presentation and in disease outcome. Phenotypic variation is a product of and provides a window into the evolutionary processes that have shaped regulatory networks. The long-term goal of this project is to understand at a mechanistic level the sources and the consequences of variation in complex phenotypes. Specifically, this project will study variation in single-cell morphology of the yeast Saccharomyces cerevisiae in different genetic backgrounds. Yeast is an established model organism for understanding basic cellular processes, and also an important model for human disease, particularly the cell-cycle defects and chromosome instability (CIN) associated with cancer. Specific Aim 1 is to determine the congruence between mechanisms that buffer complex phenotypes against environmental variation and against genetic variation. Previous experiments identified hundreds of deletion mutations that increase morphological variation in isogenic cells. This disrupted buffering of environmental differences will be compared to that of genetic differences by introducing a subset of these mutations into diverse yeast strains. Because of the importance of transcriptional networks in robustness, particular focus will be on mutations in genes that encode transcriptional regulators. Genes that act in chromosome organization are also disproportionately found to be required for buffering. One such gene, HTZ1, encodes H2A.Z, a histone variant that is required for proper transcriptional regulation and also proper chromosome segregation. Specific Aim 2 is to determine the relative contributions to phenotypic variation of impaired buffering and CIN, using engineered mutations in HTZ1 that separate these two sources of variation. Whereas the genetic variability that accompanies CIN in cancer has been a long-recognized potential source of phenotypic heterogeneity, impaired buffering of regulatory networks has not been. Specific Aim 3 is to test whether partial loss-of-function mutations in essential genes impair buffering. Nonessential genes that contribute to robustness share properties with essential genes, such as participation in core cellular processes and high connectivity in genetic networks. This observation raises the possibility that essential genes are major contributors to robustness. A comprehensive collection of strains containing hypomorphic mutations in essential genes will be used to determine the extent to which these genes buffer morphological phenotypes. The project will test key hypotheses about the genetic architecture of robustness and may reveal an underappreciated mechanism generating heterogeneity in human disease. PUBLIC HEALTH RELEVANCE: Complex human diseases, such as cancer, are affected by a large number of environmental and genetic factors, making them difficult to diagnose, treat and prevent. We will use laboratory experiments to understand how variation in these factors produces variability in complex traits. This will advance our understanding of the molecular mechanisms underlying healthy and diseased states.